Abstract

The scaffold protein synectin plays a critical role in the trafficking and regulation of membrane receptor pathways. As platelet-derived growth factor receptor (PDGFR) is essential for hepatic stellate cell (HSC) activation and liver fibrosis, we sought to determine the role of synectin on the PDGFR pathway and development of liver fibrosis. Mice with deletion of synectin from HSC were found to be protected from liver fibrosis. mRNA sequencing revealed that knockdown of synectin in HSC demonstrated reductions in the fibrosis pathway of genes, including PDGFR-β. Chromatin IP assay of the PDGFR-β promoter upon synectin knockdown revealed a pattern of histone marks associated with decreased transcription, dependent on p300 histone acetyltransferase. Synectin knockdown was found to downregulate PDGFR-α protein levels, as well, but through an alternative mechanism: protection from autophagic degradation. Site-directed mutagenesis revealed that ubiquitination of specific PDGFR-α lysine residues was responsible for its autophagic degradation. Furthermore, functional studies showed decreased PDGF-dependent migration and proliferation of HSC after synectin knockdown. Finally, human cirrhotic livers demonstrated increased synectin protein levels. This work provides insight into differential transcriptional and posttranslational mechanisms of synectin regulation of PDGFRs, which are critical to fibrogenesis.

Figure 6

(A) LOGO depicting sequence homology of PDGFR-α and PDGFR-β at known PDGFR-α ubiquitination sites (Weblogo 3.5). (B–D) LX2 cells were transduced with an adenovirus encoding a PDGFR-β mutant 606/971 flag–tagged construct or pShuttle vector, and treated overnight with bafilomycin (10 μM, B and C) in serum-starved media. Overexpression of the PDGFR-β mutant 606/971 in LX2 cells resulted in a 2-fold increase of PDGFR-β mutant after incubation with bafilomycin (B) and increased colocalization with autophagosome marker LC3b (C) and p62 (D). Two images were obtained from 3 separate wells for a total of 6 fields taken at 63×. Pearson’s coefficient was calculated by JoCIP plug-in in ImageJ for panels C and D. Scale bar: 10 μm. (E) Mutation of the PDGFR-α at K971 prevented its degradation after PDGF stimulation, n = 3. Samples were run on the same gel but in noncontiguous lanes. Data are expressed as mean ± SEM. One-way ANOVA with Bonferroni’s multiple comparison test was used to analyze groups for statistical significance (**P < 0.001, ). Student unpaired t test was used to analyze the differences between 2 groups (***P < 0.0001).